GH172 Difructose Dianhydride I Synthase: A Two‐Stepped Journey via Glycosylation and Cyclization Using QM/MM Metadynamics

ABSTRACT The difructose dianhydride I synthase/hydrolase catalyzes the reversible transformation of inulobiose into difructose dianhydride I, a component of caramel. The proposed reaction mechanism of such a transformation consists of a glycosylation step followed by a cyclization to yield the dianhydride from the disaccharide. Here, we make use of extensive hybrid QM/MM metadynamics studies to shed light on the molecular basis of it. Our simulations show that the global reaction for the transformation of inulobiose by is slightly exergonic in good agreement with the experimental data. Furthermore, we observe that the glycosylation step is the rate‐limiting step. Interestingly, our work shows that the –1 sugar of inulobiose changes from an initial E conformation into a E puckering via the conformational pathway E E / T E E . Our calculations highlight that in the cyclization step, the three residues E85, K147, and N226 are essential for the rotation of the +1 sugar in the substrate to facilitate the intramolecular attack of oxygen O1' to the anomeric carbon. Prompted by these results, we expressed and assayed the N226A variant. Enzyme activity data confirm an important role for N226 during the cyclization step but show that it is dispensable for the hydrolysis step.